BM-derived CD45⁺ cells isolated from ΔSTAT5A and ΔSTAT5B transgenic mice fail to undergo cell expansion when cultured with IL-3. (A) BM-derived CD45⁺ cells (top panel) and mature EC (bottom panel) were isolated and lysed from the independent founder lines (65 and 490) and from WT mice. Protein extracts were immunoprecipitated with the 9E10 anti-Myc antibody and subjected to SDS-PAGE, and the filters were IB with the 9E10 anti-Myc antibody. (B) Cell extracts from WT, Tie2-Δ5A–, and Tie2-Δ5B–derived ECs, treated or untreated with IL-3, were analyzed by WB using anti–p-STAT5, anti–STAT5, and 9E10 anti-Myc antibodies. (C) Hematoxylin-eosin–stained sections of Matrigel plugs containing IL-3 recovered from WT, Tie2-Δ5A, and Tie2-Δ5B mice are reported. Quantification of neovascularization was expressed as percentage (± SD) of the vessel area to the total Matrigel area. Each individual experimental group included 5 mice. (*P < .05, experimental groups vs WT.) (D) Cell extracts from IL-3–cultured-CD45⁺ cells obtained from WT, Tie2-Δ5A, or Tie2-Δ5B mice, transfected with the STAT5B or STAT5A DN constructs, respectively, were analyzed by WB using anti–p-STAT5, anti-STAT5, anti–cyclin D1, 9E10 anti-Myc, and anti–β-actin antibodies. (E) The percentage of cells in the S phase evaluated by FACS analysis on CD45⁺ cells from WT, Tie2-Δ5A, and Tie2-Δ5B mice and on Tie2-Δ5A and Tie2-Δ5B CD45⁺ cells, transfected with the STAT5B and STAT5A DN constructs, respectively (*P < .05, experimental groups vs WT). IL-3 was added in the culture media. (F) Q-RT-PCR was performed to evaluate the expression of arterial markers on CD45⁺ cells isolated from WT, Tie2-Δ5A, and Tie2-Δ5B mice. Expression levels are presented as described above. (*P < .05, experimental groups vs WT.) Three different experiments were performed with comparable results.